This project is designed to further purify and characterize various forms of glutamate decarboxylase (GAD), cysteic/cysteinesulfinic acids decarboxylase (CAD/CSAD) and serine-transhydroxymethylase (serine-THM), the biosynthetic enzymes for GABA, taurine and glycine respectively, and to delineate the role of these enzymes in the regulation of the effective levels of GABA, taurine and glycine in connection with their important roles as neurotransmitters or modulators in mammalian central nervous system. The purified enzyme preparations will also serve as antigens for the production of specific antibodies, so that the precise cellular and subcellular location of these enzymes and their interrelationship can be visualized by immunocytochemical methods. Double-staining techniques will be used to localize the neuronal and the glial GAD so that the relationship of glia and neuron and the role of glia in the GABA system can be better understood. More specifically, I plan to perform the following studies: (1) purify neuronal GAD, CAD/CSAD and serine-THM from gray matter of bovine brain and glial GAD from glia-enriched fraction or from non-neural tissues, e.g., heart and kidney with methods similar to those I have developed for successful purifications of GAD and GABA-transamine from mouse brain, GAD and CAD from bovine brain and choline acetyltransferase from Torpedo (2) localize these enzymes on tissue section by immunocytochemical methods which we have successfully applied to the localization of GAD in various regions of CNS in rat and rabbit (3) perform immunochemical studies to determine species-, tissue-, and cell specificities of these enzymes. Radioimmuno- or enzymeimmuno assays will be developed so that the actual quantities of the enzyme proteins can be accurately determined in addition to the enzyme activities, (4) perform developmental studies with a view to correlating biochemical, immunochemical and morphological changes during maturation. The Preliminary Results, which show that GAD and CAD have been purified from bovine brain and specific antibodies are being produced, combined with the experience of the Principal Investigator have shown the feasibility of the proposed project.